Isotope dilution species-specific method

Uncontrolled species transformations during analysis form another source of error. For methylmercury determinations in sediments it was demonstrated that errors of up to 80% resulted from the formation of the compound from inorganic mercury during separation and analysis [28, 29], For the study of possible species transformations during analysis multiple isotope dilution could be used as a diagnostic tool for identifying the error and bias inherent in specific methods of storage, sample preparation and measurement [30, 31]. 

F. Yang, R. E. Sturgeon, S. McSheehy, Z. Mester, Comparison of extraction methods for quantitation of methionine and selenomethionine in yeast by species specific isotope dilution gas chromatography-mass spectrometry, J. Chromatogr. A, 1055 (2004), 177-184. 

Dimethyl-Tl (Me2Tl ) was determined in 500 ml sea water samples by combining Isotope Dilution Mass Spectrometry (IDMS) with a species-specific extraction method (18). A ° Tl-enriched spike solution was applied for the isotope dilution step and the production of positive thermal ions was used for mass spectrometric measurements. After species-unspecific T1 enrichment at an anion exchanger, inorganic T1 was oxidized to Tl(III) by a Br2 solution and then separated by extraction with methyl xo-butyl ketone, whereas Me2Tl remained in the aqueous phase. The detection limit for Mc2Tl is 440 pg 1 (as Tl). 

Recent clearance studies use stable isotope dilution methods. MS methods make it possible to measure deuterated and endogenous species simultaneously and specifically. LC-MS methods offer advantages over GC-MS methods, such as streamlined sample preparation (no derivatization necessary), high recovery and sensitivity and superior specificity. 

With radioisotopes now available for many elements, the tracer technique became generally applicable. New variants were developed, such as neutron activation analysis, which was introduced in 1936 for the determination of dysprosium in rare-earth samples (Hevesy and Levi 1936) and subsequently became a widely used technique for sensitive trace analyses, particularly when much larger neutron fluxes became available with the advent of nuclear reactors. Another frequently applied method for trace determination is isotope dilution the species to be determined in the sample is diluted by addition of a known amount of the same species labeled with known specific activity. From the specific activity then resulting and measured, the original quantity of the species is derived, even if only a fi action of the species is finally recovered. The impact on biosciences was revolutionary, when suitable isotopes of key elements in the biosphere were soon discovered (Ti/2 = 10 min) was one of I. Curie and... 

Hyphenated techniques are the analytical methods most frequently used today for elemental speciation. For that purpose, a separation technique, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), or gel electrophoresis (GE), is coupled on-line with an elemental detection method, such as ICP-MS [67]. Hyphenated ICP-MS techniques can also be combined with the isotope dilution method for quantification of elemental species. Two different spiking modes are possible, the species-specific and spedes-unspedfic mode. Rottmann and Heumann were the first to present a setup for H PLC-ICP-IDMS enabling the use of these two spiking modes [68] (Figure 8.14). 

These methods have been widely used. Isotopic dilution methods are based on the dilution of the biotin to be estimated by a known amount of labeled biotin, followed by the determination of the specific activity of the avidin complexed or the free biotin after thorough separation of both species. [ ClBiotin (78,79), [ Hjbiotin (80), and I-labeled derivatives (Fig. 12) of biotin (81) have been used. The separation of free and avidin-complexed biotin has been achieved with bentonite (80), zinc hydroxide (78), dextran-coated charcoal (79), polyethyleneglycol (82), antibodies (83) or by the use of covalently linked avidin on cellulose (84). The sensitivity depends on the specific activity of the labeled biotin and can range from 20 ng with [ Cjbiotin, to 1 ng with [ Hjbiotin, down to 1 pg with I-labeled derivatives. 

Chapters on sample introduction and hyphenated sample treatment and ICP systems have also been further updated since the last edition. No doubt that chromatographic, electrophoresis, flow injection and field flow fraction separations have extended ICP-MS (and AES) measurements as the mainstay of elanental specia-tion measurements in biological and environmental fields. Without the combination of these separation techniques and ICP measurements, elemental speciation applications would be severely hampered... if not impossible (Chapter 18). The ability to measure P and S with high sensitivity has opened up new opportunities in proteomics, for example. Species-specific and unspecific isotopic dilution (ID-MS) has been critical in quantifying speciation analysis and revealing recovery errors (Chapter 13). Species-specific techniques have been applied to identify species transformations, resulting in the development of multi-species methods whereas, hyphenated species-unspecific ICP-ID-MS determinations of heteroatoms such as sulfur have become a common quantification technique in proteomics. 

The main challenge when applying this method for protein analysis is the lack of commercial isotopically labeled proteins. Thus, most applications are focused on small molecules, such as organic mercury, organic tin and so on. However, there is increasing interest in the use of the ICP-MS linked system and species-specific isotope dilution for quantification of peptides or proteins due to the outstanding performance of ICP-MS. 

Encinar et al. developed a method for the accurate determination of sele-noamino acids in human serum by species-specific isotope dilution analysis. A human serum was enzymatically digested, and then the selenoamino acid and carboxymethylated selenocysteine were separated and quantified by HPLC-ICP-MS. Quantification of selenomethionine was carried out by isotope dilution using a synthetic Se-labeled counterpart. The selenomethionine in samples was also measured by using selenomethionine as an internal standard. The instrumental detection limit was down to 75 fg Se and the precision was better than 5% RSD. ... 

Later, Busto et al. used synthesized Fe-labeled transferrin to determine individual transferrin isoforms in human serum samples for the species-specific mode. The stability of the prepared proteins was tested for 1 week and no iron exchange had occurred. They concluded that the results are in good agreement with other calibration methods, e.g. the species-unspecific method however, the species-spedfic mode can offer better precision. Hoppler et al. also synthesized and characterized Fe-labeled Phaseolus vulgaris ferritin for isotope dilution analysis. " ... 

This technique has been used for a variety of element species, such as trimethyl- and triethyl-lead, organotins, iodine, methylmercury, and chromium. With HPLC, isotope dilution has been performed with species-specific as well as species-unspecific methods. 

The data evaluation for species-specific isotope dilution is achieved by peak area determination of the respective isotope peaks. A detailed description of the data evaluation in species-specific isotope dilution methods with ICP-MS detection can be found in Section 7.5.5. In species-specific isotope dilution procedures, isotopically labelled species are required, and the preparation of isotopically labelled methylmercury and butylated tin species is also described in detail in Section 7.5.5. 

The calculated mass bias factor a is then applied to the actual measurements and used for correction of the isotope ratio value. Simultaneous methods have been developed by determination of the mass bias factor on a different element, for example thallium for the mass bias correction of lead and mercury.This principle has been adopted for species-specific isotope dilution measurements with GC-ICP-MS, with a system enabling the simultaneous aspiration of a standard solution during GC introduction. The simultaneous method was compared with mass bias correction using additionally measured standards, and showed satisfactory accuracy. Thallium was used for mass bias correction of lead and mercury species while antimony has been used for correcting tin species isotope ratios. ... 

For mercury species determination in natural gas condensate, isotopically enriched Mc2Hg, MeHgCl and HgCh were prepared and used for species-specific isotope dilution and method assurance tests. ° This method gave excellent detection limits and improved matrix tolerance compared with other speciation approaches for samples in an organic matrix. 

A modification of this method was developed for the simultaneous determination of MBT, DBT and TBT in coastal seawater samples, based on the application of species-specific isotope dilution with a Sn-enriched spike of the three butyltin compounds of interest... 

Yang et al. described the development of a sensitive method for the accurate and precise quantitative determination of TBT and DBT in sediments by species-specific isotope dilution ICP-MS. Using GC for sample introduction and analyte separation, detection with quadrupole ICP-MS and sector-field ICP-MS were compared. A more than two-fold improvement in precision of calculated °Sn/ Sn ratios was obtained for both TBT and DBT in standards using GC-ICP-SF-MS as compared to GC coupled with quadrupole ICP-MS. Superior limits of detection were obtained for SF-ICP-MS coupling because of the improved signal-to-background ratio. 

The only respect in which the hot atom chemistry of organometallic compounds has so far been applied to other fields of study is in the area of isotope enrichment. Much of this has been done for isolation of radioactive nuclides from other radioactive species for the purpose of nuclear chemical study, or for the preparation of high specific activity radioactive tracers. Some examples of these applications have been given in Table II. The most serious difficulty with preparation of carrier-free tracers by this method is that of radiolysis of the target compound, which can be severe under conditions suited to commercial isotope production, so that the radiolysis products dilute the enriched isotopes. A balance can be struck in some cases, however, between high yield and high specific activity (19, 7J),... 

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